Biomarkers of Oxidative Stress and Their Clinical Relevance in Type 2 Diabetes Mellitus Patients: A Systematic Review

Assessing oxidative stress is vital in managing type 2 diabetes mellitus (T2DM) and its complications. This systematic review aims to identify the most important oxidative stress markers in T2DM patients and predict associated complications. A literature search was conducted from 2013 to 2023, focusing on case-control, cohort, cross-sectional, and randomized control trials. The included studies had open access and scientific methodologies for assessing oxidative stress markers, while the excluded studies were not published in English or lacked primary objectives related to oxidative stress markers and T2DM or its complications. The quality of eligible studies was evaluated using the Newcastle-Ottawa Scale (NOS) for case-control, cohort, and cross-sectional studies and the Jadad Scale for RCTs. Eighteen studies were selected for the review and 25 potential markers like malondialdehyde (MDA), 11 thiobarbituric acid reactive substances (TBARS), 8-hydroxydeoxyguanosine (8-OHdG), glutathione (GSH), superoxide dismutase (SOD), and isoprostanes were found to be the most commonly used markers to assess oxidative stress in T2DM. These markers help to assess oxidative stress levels in T2DM individuals as well as correlate with diabetic complications. Therefore, assessment and understanding of the role of oxidative stress in T2DM pathophysiology are crucial for improving patient care and mitigating complications.


Introduction And Background
Oxidative stress is an imbalance between reactive oxygen species (ROS) and antioxidant defense mechanisms in the body.Chemically, ROS is a byproduct formed from hydrogen peroxide (H2O2), oxygen (O2), and water (H2O) [1,2].In the human body, endogenous ROS are produced mainly from mitochondria as a byproduct of mitochondrial respiration [3].
Excessive ROS in the body exhausts cellular antioxidant defense mechanisms, leading to oxidative stress.This dysregulation of redox homeostasis has been linked to the pathogenesis of aging and various diseases, including diabetes [4,5].Type 2 diabetes mellitus (T2DM) is a significant global health challenge, and its prevalence is increasing worldwide [6,7].Studies have shown that oxidative stress is a key contributor to the pathogenesis and progression of T2DM and its associated complications [8].
These complications can be due to the damage caused by reactive oxygen species to mitochondrial DNA, proteins, and lipids [9].In the body, ROS oxidize proteins, leading to altered protein structure and function, which alter their active sites and enzymatic activities, contributing to different disease conditions [10,11].By directly attacking DNA, ROS can cause base modifications, strand breaks, and chromosomal rearrangements, leading to mutations and cell death and being an important cause of oncogenesis, which is, unfortunately, an under-recognized complication in long-standing diabetes [12][13][14].Recent studies have increasingly recognized oxidative stress as a key contributor to the development and progression of diabetes and its complications.With the present evidence linking oxidative stress and T2DM pathology, there is a growing demand to identify potential biomarkers of oxidative stress for early detection, risk mitigation, and monitoring of patient responsiveness to medications.
The current systematic review aims to provide a qualitative summary of the diagnostic utility and predictive value of oxidative stress markers in the complications of T2DM.Given the abundance of literature on oxidative stress markers and diabetes, it is imperative to derive a qualitative analysis of the published literature to identify the most effective oxidative stress markers in T2DM.Therefore, this systematic review attempts to address the existing knowledge gaps and find the best oxidative stress markers in diabetic mellitus and its complications.

Review Materials and methods
For this systematic review, we adhered to the Preferred Reporting Items for Systematic Reviews (PRISMA) Updated Guidelines 2020 [15].The article selection process is comprehensively illustrated in the PRISMA flowchart (Figure 1).

FIGURE 1: PRISMA flowchart explaining the selection of studies for systematic review.
Reference [15].

Search Strategy
A detailed literature search was conducted across scholarly databases such as PubMed, Google Scholar, and the Cochrane Library, with a focus on published studies between 2013 and 2023.Only case-control, cohort, cross-sectional, and randomized control studies employing systematic methodology for measuring oxidative stress were considered for review, and a detailed process of exclusion and inclusion was employed to filter the studies.Boolean operators such as 'AND' and 'OR' were utilized in the search, incorporating MeSH terms such as ('biomarker' OR 'biological marker' OR 'biomolecular marker' OR 'bioindicator*' OR 'biomolecular indicator') AND ('oxidative stress' OR 'oxidative damage' OR 'reactive oxygen species' OR 'ROS' OR 'oxidative biomarker' OR 'oxidative marker') AND ('type 2 diabetes mellitus' OR 'T2DM' OR 'diabetes mellitus, type 2' OR 'insulin dependent diabetes mellitus').The search was limited to published, open-access studies from the last 10 years.

Eligibility Criteria
The eligibility criteria for this systematic review were precisely defined to ensure the inclusion of scientifically sound and relevant studies.The review considered only case-control, cohort, cross-sectional, and randomized control studies.Only fully open-access studies published between 2013 and 2023 were included.Studies that used detailed and scientific methodologies for oxidative stress marker assessment were included.Conversely, we excluded studies published in languages other than English and studies that did not assess oxidative stress markers or T2DM or its complications as a primary objective.Conference presentations were also excluded.

Data Management
Data extraction and the preliminary reading of the study titles and abstracts were performed independently by the first, second, and third authors.The fourth and fifth authors then read the full text to check for eligibility.Any discrepancies that arose were resolved through discussion or consultation with the other authors.The data extracted from each eligible study were tabulated and included information such as the first author, year, study title, study design, sample size, oxidative stress markers analyzed, and conclusions of the study.

Quality Assessment
The quality of the selected studies was independently assessed by all authors using the Newcastle-Ottawa Scale (NOS) for case-control, cohort, and cross-sectional studies.The Jadad scale was used for randomized controlled trials (RCTs).The NOS for case-control and cohort studies evaluated criteria such as the adequacy of the case definition, representativeness of cases, selection and definition of controls, comparability of cases and controls, ascertainment of exposure, and non-response rate.
For cross-sectional studies, the NOS quality evaluation assessed the representativeness of the sample, sample size, selection of study subjects, comparability between groups, ascertainment of exposure, assessment of outcome, and description of statistical tests.Quality evaluation with the Jadad scale for RCTs assessed randomization, blinding, and handling of withdrawals and dropouts.

Study Characteristics
A total of 18 studies were selected for final review.The selected studies consisted of 11 case-control studies, 1 cohort study, 3 cross-sectional studies, and 3 randomized controlled trials.The characteristics of each study, including study design, sample size, oxidative stress markers assessed, and key findings, are summarized in Table 1.

Discussion
There are diverse methodologies employed by researchers for assessing oxidative stress markers.This oxidative stress assessment is highly important for the treatment of various diseases.They can act as crucial indicators of cellular damage and provide information about the pathophysiological mechanisms of diseases such as diabetes, cancer, and metabolic syndromes.Several mechanistic and epidemiological studies point to the connection between diabetes and cancer through ROS [35].
Type 2 diabetes is a chronic metabolic disorder characterized by insulin resistance and relative insulin deficiency [34].Oxidative stress plays a significant role in the pathophysiology of T2DM, contributing to its development and progression, as well as the associated complications [36].Several oxidative stress markers have been associated with T2DM.The important marker discussed in the majority of the studies is MDA, which is a byproduct of lipid peroxidation that is frequently elevated in T2DM patients and indicates increased oxidative damage to lipids, which contributes to the development of complications.Furthermore, alterations in antioxidant defense mechanisms, such as decreased activity of SOD, catalase (CAT), and glutathione peroxidase (GPx), are frequently reported in T2DM patients, further exacerbating oxidative stress.
The most important markers of oxidative stress identified through the review are discussed in brief below.

MDA
Malondialdehyde is a product of polyunsaturated fatty acid peroxidation.Lipid peroxidation occurs when free radicals attack the carbon-carbon double bond of lipids.An increase in free radicals in the body is reflected by an increase in MDA levels [37].Although there are variations in the levels of MDA in different samples, MDA is considered a reliable marker of oxidative stress under various disease conditions [38].The thiobarbituric acid (TBA) assay is commonly employed to measure MDA in biological samples.Due to the instability of MDA in biological samples and the lack of specificity of the TBA reaction with MDA, total TBARS are measured as an oxidative stress biomarker in many studies [38].

SOD
Superoxide dismutase is an essential metalloenzyme found in living organisms.It serves as the first line of defense against reactive oxygen species.There are various isoforms of SOD based on their metal cofactors, as described in previous studies [39].SOD can spontaneously dismutate superoxide radicals and cleave hydrogen peroxides and hydroperoxides to create stable molecules by undergoing the oxidation-reduction of metal ions [40].

CAT
Catalase is another key enzyme in the first line of defense against reactive oxygen species and is an important NADPH-binding protein [41].It protects the body from oxidative damage by converting hydrogen peroxide into water and oxygen.CAT and SOD are enzymes involved in the same pathway of free radical mitigation [40][41][42][43].

GSH
The ratio of reduced GSH to GSSG is an indicator of cellular oxidative stress levels [44].Glutathione disulfide is an important redox couple in cells, and its deficiency causes oxidative stress in the body.Hence, GSH is involved in the pathogenesis of various diseases [45].

Isoprostanes
These are a type of prostaglandin-like compound formed independently of the cyclooxygenase pathway and generated by the nonenzymatic peroxidation of PUFAs, especially arachidonic acid [45].Arachidonic acid peroxidation produces three arachidonoyl radicals that undergo endocyclization to form four PGH2-like bicyclic endoperoxides [46].Under oxidative stress, a fraction of lipid hydroperoxides undergo rearrangement and cyclization reactions to form isoprostanes [45,47].Isoprostanes are stable compounds that can be detected in various biological samples, such as urine, plasma, serum, and tissues [45].

GPx
Glutathione peroxidase utilizes GSH as a cofactor in its catalytic cycle.In the presence of reduced GSH, GPx catalyzes the reduction of peroxides to their corresponding alcohols, forming oxidized glutathione (GSSG) in the process.The reaction mechanism typically involves the transfer of electrons from GSH to the peroxide substrate, resulting in the formation of water or alcohol and the oxidation of GSH to GSSG [50,51].
The formation of 8-OHdG leads to mutations in the DNA sequence if not repaired spontaneously.The accumulation of 8-OHdG in the genome is associated with various pathological conditions [55].Apart from other oxidized guanine molecules, 8-OHdg can cross the cell membrane.Hence, the presence of 8-OHdg can be detected in bodily fluids such as urine and serum, and 8-OHdg can be used as a sensitive marker for oxidative stress assessment [56].Measurement of 8-OHdG in urine and serum samples can indicate the progression and development of complications in diseases such as diabetes, cancer, and some bacterial infections [22,32,57,58].
Although we followed a strict systematic methodology to filter out the best oxidative stress markers, there are some limitations in the current review that need to be considered.First, the heterogeneity among studies in terms of sample size, study design, and methodology may introduce variability in the findings, which limits the generalizability of the results.Additionally, many of the reviewed studies relied on cross-sectional and case-control study designs, which prevent the establishment of causal or temporal relationships between oxidative stress markers and diabetic complications.Therefore, longitudinal studies with larger cohorts are necessary to validate the predictive utility of these markers and better understand their role in disease progression.

Conclusions
This systematic review provides a qualitative summary of oxidative stress markers to predict complications in type 2 diabetes patients.Lipid peroxidation is the most common indicator of oxidative damage and is measured by assays such as TBARS and MDA levels.The levels of specific oxidative stress biomarkers, such as 8-OHdG, can be measured in both urine and serum.These markers provide insights into pathophysiological mechanisms and serve as potential prognostic indicators for disease progression and therapeutic response.Measuring oxidative stress markers can help mitigate complications in T2DM.The majority of the studies measured MDA as an oxidative stress marker, followed by SOD> Isoprostanes>GSH>TAC>Gpx>CAT>TOS>TAS>TBARS.Other important markers of oxidative stress measurement were LPO, NO, HO, POVPC, PGPC, AOPP, IMA, OSI, PTGS2, protein carbonyls, Ox-LDL, AGES, and sORP.

FIGURE 2 :
FIGURE 2: Bar chart depicting the frequency of different oxidative stress markers analyzed in studies reviewed, highlighting the number of studies conducted for each marker.MDA was the most common marker used by majority of the studies.